Oncogene (2014) 33, 3422–3431 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE Targeting the MUC1-C oncoprotein downregulates HER2 activation and abrogates trastuzumab resistance in breast cancer cells

D Raina1, Y Uchida2, A Kharbanda2, H Rajabi2, G Panchamoorthy1, C Jin2, S Kharbanda1, M Scaltriti3, J Baselga3 and D Kufe2

Patients with HER2-positive breast cancer often exhibit intrinsic or acquired resistance to trastuzumab treatment. The transmembrane mucin 1 (MUC1) oncoprotein is aberrantly overexpressed in breast cancer cells and associates with HER2. The present studies demonstrate that silencing MUC1 C-terminal subunit (MUC1-C) in HER2-overexpressing SKBR3 and BT474 breast cancer cells results in the downregulation of constitutive HER2 activation. Moreover, treatment with the MUC1-C inhibitor, GO-203, was associated with disruption of MUC1–C/HER2 complexes and decreases in tyrosine-phosphorylated HER2 (p-HER2) levels. In studies of trastuzumab- resistant SKBR3R and BT474R cells, we found that the association between MUC1-C and HER2 is markedly increased (B20-fold) as compared with that in sensitive cells. In addition, silencing MUC1-C in the trastuzumab-resistant cells or treatment with GO-203 decreased p-HER2 and AKT activation. Moreover, targeting MUC1-C was associated with the downregulation of phospho-p27 and cyclin E, which confer trastuzumab resistance. Consistent with these results, targeting MUC1-C inhibited the growth and clonogenic survival of both trastuzumab-resistant cells. Our results further demonstrate that silencing MUC1-C reverses resistance to trastuzumab and that the combination of GO-203 and trastuzumab is highly synergistic. These findings indicate that MUC1-C contributes to constitutive activation of the HER2 pathway and that targeting MUC1-C represents a potential approach to abrogate trastuzumab resistance.

Oncogene (2014) 33, 3422–3431; doi:10.1038/onc.2013.308; published online 5 August 2013 Keywords: MUC1; HER2; HER3; trastuzumab; resistance; breast cancer

INTRODUCTION that resistance of HER2-overexpressing breast cancer cells to The HER2/ERBB2 receptor tyrosine kinase (RTK) is overexpressed in trastuzumab is conferred by (i) upregulation of cyclin E and an approximately 20% of human breast cancers and is associated increase in CDK2 activity,20 and (ii) decreased expression of the with aggressive disease and poor survival.1,2 HER2 forms PPM1H phosphatase that regulates stability of the CDK inhibitor heterodimers with HER3 and thereby phosphorylates and p27.21,22 These findings have provided the experimental basis for activates HER3.3,4 Trastuzumab is a humanized monoclonal designing strategies that target pathways associated with antibody that binds to the HER2 extracellular domain and trastuzumab resistance to reverse unresponsiveness to this agent. destabilizes ligand-independent HER2/HER3 complexes.5 Mucin 1 (MUC1) is a heterodimeric protein that associates with Targeting of HER2 with trastuzumab in HER2-overexpressing HER2 at the surface of breast cancer cells.23,24 MUC1 is translated breast cancer cells also decreases HER2 levels6,7 and induces G1 as a single polypeptide that undergoes autocleavage into arrest by stabilizing the CDK inhibitor p27.8 Trastuzumab extends N-terminal (MUC1-N) and C-terminal (MUC1-C) fragments, which the overall survival of certain patients with HER2-overexpressing in turn form a stable complex at the cell membrane.24,25 The breast cancers when used as monotherapy or in combination with MUC1-N/MUC1-C heterodimer is positioned at the apical border of chemotherapy.9–11 However, many patients exhibit de novo breast epithelial cells and is sequestered from RTKs that are unresponsiveness to trastuzumab or develop acquired resistance expressed at the basolateral membranes.24,25 However, with loss after treatment.11 Trastuzumab resistance has been associated of apical–basal polarity as a result of stress or transformation, with constitutive activation of the phosphatidylinositol-3 kinase MUC1 is repositioned over the entire cell membrane and interacts pathway as a result of phosphatase and tensin homolog (PTEN) with RTKs such as HER2.23–25 MUC1-N, the mucin component of deficiency12 or PIK3CA gene mutations.13 Phosphatase and tensin the heterodimer, is shed from the cell surface.24,25 The MUC1-C homolog has also been linked to SRC activation and thereby subunit spans the cell membrane and includes a 58-amino-acid trastuzumab resistance in breast cancer cells and in breast (aa) extracellular domain, a 28-aa transmembrane domain and a tumors.14 Additional mechanisms of resistance have included 72-aa cytoplasmic domain (CD). MUC1-C associates in part with expression of a truncated p95HER2 that lacks the trastuzumab- RTKs through extracellular galectin-3 bridges.26 In addition, the binding domain,15 heterodimerization with other RTKs16–18 and MUC1-C-CD functions as a substrate for phosphorylation by RTKs downregulation of HER2 expression.19 Other studies have shown and SRC.24,25 The MUC1-C-CD also contains a YHPM motif that,

1Genus Oncology, Boston, MA, USA; 2Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA and 3Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Correspondence: Dr D Kufe, Medical Oncology, Dana-Farber Cancer Institute, Harvard, Medical School, 450 Brookline Avenue, Dana 830, Boston, MA 02215, USA. E-mail: [email protected] Received 10 April 2013; revised 24 June 2013; accepted 25 June 2013; published online 5 August 2013 MUC1-C promotes HER2 activation and trastuzumab resistance D Raina et al 3423 when phosphorylated on tyrosine, functions as a binding site for performed on SKBR3 and BT474 breast cancer cells that phosphatidylinositol-3 kinase p85 SH2 domains.27 These findings overexpress HER2 and are dependent on phosphorylated HER2 and the demonstration that MUC1-C overexpression is sufficient to (p-HER2) for growth and survival.3 Levels of HER2 and MUC1-C induce anchorage-independent growth and tumorigenicity28,29 were found to be similar in these cells (Figure 1a). Co- provided the basis for developing agents that block the MUC1-C- immunoprecipitation studies further demonstrated that MUC1-C transforming function.25 In this way, the MUC1-C-CD contains a associates with HER2 in both SKBR3 and BT474 cells (Figure 1b). To CQC motif that is necessary for its homodimerization and assess the potential effects of MUC1-C on HER2 signaling, we function.30 Notably, cell-penetrating peptides that bind to the stably silenced MUC1-C in SKBR3 cells (Figure 1c, left). Notably, MUC1-C CQC motif are effective in inhibiting growth and inducing MUC1-C silencing was associated with the downregulation of death of human breast cancer cells growing in vitro and as p-HER2, but not HER2, abundance, consistent with a decrease in xenografts in mice.31 However, the effects of MUC1-C inhibition on HER2 activation (Figure 1c, left). In concert with these results, (i) the interaction between MUC1-C and HER2, and (ii) HER2 silencing MUC1-C in BT474 cells similarly resulted in suppression signaling in breast cancer cells are not known. of p-HER2 levels (Figure 1c, right). MUC1-C silencing was also The present studies demonstrate that MUC1-C contributes to associated with a decrease in SKBR3 cell growth (Figure 1d, left) HER2 activation in HER2-overexpressing breast cancer cells and and BT474 cell growth (Figure 1d, right). Moreover, colony thereby promotes their growth and clonogenic survival. The formation was substantially decreased by silencing MUC1-C results also demonstrate that the formation of MUC1-C/HER2 expression in both SKBR3 (Figure 1e, left) and BT474 (Figure 1e, complexes is substantially increased in the setting of trastuzumab right) cells. These findings indicated that MUC1-C contributes to resistance and that targeting MUC1-C in trastuzumab-resistant HER2 activation and proliferation of HER2-overexpressing breast cells results in the downregulation of HER2 activation. In concert cancer cells. with these findings, we show that targeting MUC1-C is effective in reversing trastuzumab resistance. The MUC1-C inhibitor, GO-203, downregulates HER2 phosphorylation The 72-aa MUC1-CD contains a CQC motif that is necessary for its RESULTS homodimerization30,32 (Figure 2a). The results of pull-down studies Silencing MUC1-C suppresses HER2 activation using lysates from SKBR3 cells demonstrated that MUC1-CD is MUC1 associates with HER2 in non-HER2-amplified breast cancer sufficient for forming complexes with HER2 (Figure 2a, left). cells and this interaction is increased by heregulin stimulation.23 However, binding to HER2 was not detectable with MUC1-CD in However, the functional significance of the MUC1-C/HER2 which the CQC motif had been mutated to AQA (Figure 2a, left). interaction has remained unclear. Accordingly, studies were Similar results were obtained when pull-down experiments were

Figure 1. Silencing MUC1-C downregulates HER2 activation and colony formation. (a) Lysates from SKBR3 and BT474 cells were immunoblotted (IB) with the indicated antibodies. (b) Lysates from SKBR3 (left) and BT474 (right) cells were precipitated with anti-MUC1-C or a control immunoglobulin G (IgG). The precipitates were immunoblotted with the indicated antibodies. (c). SKBR3 (left) and BT474 (right) cells were infected with lentiviruses to stably express a control shRNA (CshRNA) or a MUC1shRNA. Lysates were immunoblotted with the indicated antibodies. (d) The indicated SKBR3 (left) and BT474 (right) cells were seeded at 5 Â 104 cells per well. The results (mean±s.d. of three replicates) are expressed as the cell number on day 4. (e) The indicated SKBR3 (left) and BT474 (right) cells were seeded at 2000 and 1000 cells per well, respectively, and incubated for 12 days. Colony number is expressed as the mean±s.d. of three replicates. IP, immunoprecipitated.

& 2014 Macmillan Publishers Limited Oncogene (2014) 3422 – 3431 MUC1-C promotes HER2 activation and trastuzumab resistance D Raina et al 3424 performed with lysates from BT474 cells (Figure 2a, right), MUC1-C in SKBR3R and BT474R cells was associated with indicating that the MUC1-C cysteine residues are of importance decreases in p-HER3 levels (Figure 5a, left and right). Scanning of for forming MUC1-C/HER2 complexes. GO-203 is a cell-penetrating the p-HER3 signals from multiple experiments demonstrated a peptide that contains a poly-Arg cell transduction domain linked decrease in p-HER3 abundance of 55% and 52% in the SKBR3R and to CQCRRKN that binds to the MUC1-C-CD at the CQC motif and BT474R cells, respectively (Supplementary Figures S1C and D). HER3 thereby blocks MUC1-C homodimerization27,31,32 (Figure 2b). is as crucial as HER2 in promoting proliferation of breast cancer cells Another cell-penetrating peptide, designated CP-2, was synthe- that overexpress HER2.34 In that sense, the suppression of both sized in which the cysteine residues are altered to alanines, phosphorylated HER2 (Figure 4a and b) and HER3 (Figure 5a) was resulting in an inactive control that does not inhibit MUC1-C associated with a marked decrease in colony formation (Figure 5b, homodimerization27,31,32 (Figure 2b). Treatment of SKBR3 cells left and right). GO-203 treatment of SKBR3R cells also suppressed with GO-203 disrupted the interaction between MUC1-C and HER2 p-HER3 abundance and activation of the downstream effector AKT (Figure 2b, left). By contrast, CP-2 had no apparent effect on the (Figure 5c). AKT phosphorylates the CDK inhibitor p27 on T198 and abundance of MUC1-C/HER2 complexes (Figure 2b, left). GO-203, thereby inactivates p27 by preventing its localization to the but not CP-2, also blocked the interaction between MUC1-C and nucleus.35,36 In concert with the GO-203-induced decreases in AKT HER2 in BT474 cells (Figure 2b, right). Analysis of p-HER2 levels activation, phosphorylation of p27 was also decreased in the further demonstrated that GO-203, but not CP-2, decreases HER2 absence of apparent changes in p27 abundance (Figure 5c). activation in SKBR3 cells (Figure 2c, left and right). BT474 cells also Trastuzumab resistance is associated with increases in p27 responded to GO-203, and not CP-2, exposure with decreased phosphorylation22 and upregulation of cyclin E.20 In addition to HER2 activation (Figure 2d). In concert with these results, GO-203 the decreases in phospho-p27 levels, we also found that GO-203 treatment of SKBR3 (Figure 2e) and BT474 (Figure 2f) cells was treatment is associated with the downregulation of cyclin E associated with inhibition of growth. These findings and those abundance (Figure 5c). Moreover, GO-203 treatment of SKBR3R obtained with MUC1-C silencing provided support for the premise cells was associated with inhibition of growth (Figure 5d). Similar that binding of MUC1-C and HER2 contributes to HER2 activation. effects of GO-203 treatment were obtained in BT474R cells with decreases in phospho-p27 and cyclin E levels (Figure 5e) and an MUC1-C/HER2 complexes are upregulated in association with arrest of growth in vitro (Figure 5f), and as tumor xenografts in nude trastuzumab resistance mice (Figure 5g). These findings thus indicate that silencing MUC1-C Trastuzumab-resistant SKBR3R cells were generated by chronic or inhibition of MUC1-C with GO-203 results in the downregulation exposure to increasing concentrations of trastuzumab for over 18 of HER3 activation and suppression of cell growth and survival. months in vitro.33 Thus, in contrast to parental SKBR3 cells, growth of the resistant SKBR3R cells was not inhibited by exposure to Targeting MUC1-C with silencing or GO-203 treatment reverses 80 nM trastuzumab (Figure 3a). Levels of HER2 and MUC1-C were trastuzumab resistance similar in SKBR3 and SKBR3R cells (Figure 3b). Strikingly, however, MUC1-C/HER2 complexes were 19-fold higher (as determined by The demonstration that MUC1-C promotes HER2 signaling densitometric scanning of the signals) in the resistant SKBR3R cells invoked the possibility that targeting MUC1-C might affect the (Figure 3c). Analysis of trastuzumab-resistant BT474R cells33 response to trastuzumab. In this context, analysis of SKBR3R cells (Figure 3d) also demonstrated similar levels of MUC1-C and demonstrated that silencing MUC1-C is associated with increased HER2 as compared with that in the sensitive BT474 cells sensitivity to the growth inhibitory effects of trastuzumab (Figure 3e). Moreover, BT474R cells exhibited a substantial (Figure 6a). Trastuzumab resistance of BT474R cells was also increase (28-fold) in MUC1-C/HER2 complexes (Figure 3f), indicat- reversed in part by silencing MUC1-C (Figure 6b), indicating that ing that the interaction between MUC1-C and HER2 is increased in targeting MUC1-C might be effective in combination with the setting of trastuzumab resistance. trastuzumab. To assess the effects of combining GO-203 and trastuzumab, we first identified the half-maximal inhibitory concentrations (IC s) for each agent against SKBR3 and BT474 Targeting MUC1-C suppresses HER2 activation in 50 trastuzumab-resistant cells cells. In studies with SKBR3 cells, the IC50s for GO-203 and trastuzumab were 5.9 mM and 93 nM, respectively (Supplementary As found in wild-type SKBR3 cells, stable silencing of MUC1-C in Table 1A). Based on the Chou-Talalay method, we then evaluated SKBR3R (Figure 4a) and BT474R (Figure 4b) cells was associated the effects of combining GO-203 and trastuzumab. As shown in with decreases in p-HER2 levels, indicating that MUC1-C also the dose–effect curves, treatment of SKBR3 cells with the contributes to HER2 activation in trastuzumab-resistant cells. combination was more effective in inhibiting growth and survival Densitometric scanning of the p-HER2 signals from multiple than that obtained with either agent alone (Figure 6c, left). The experiments demonstrated a decrease in p-HER2 abundance of median–effect analysis and calculation of the median dose values 74% and 45% in the SKBR3R and BT474R cells, respectively further showed a B3-fold reduction in the median dose for GO- (Supplementary Figure S1A and B). In studies with SKBR3R cells, 203 in the presence of trastuzumab as compared with GO-203 we found that treatment with GO-203 is associated with alone (Figure 6c, right). Calculation of the combination index (CI) disruption of MUC1-C/HER2 complexes (Figure 4c). Similar results at the ED50, ED75 and ED90 for these agents demonstrated a high were obtained when BT474R cells were treated with the MUC1-C degree of synergy with the values o1 (Supplementary Table 1B). inhibitor (Figure 4d). GO-203 also suppressed HER2 activation in Comparable results were obtained when BT474 cells were treated both SKBR3R (Figure 4e) and BT474R (Figure 4f) cells. These results with GO-203 and trastuzumab as shown in the dose–effect curves indicate that targeting MUC1-C with silencing or GO-203 (Supplementary Figure S2A, left) and the median–effect plots treatment results in the suppression of HER2 activation in (Supplementary Figure S2A, right), and as supported by CI values trastuzumab-resistant cells. ranging from 0.47 to 0.68 (Supplementary Table 1B). IC50s for GO- 203 were also generated for the SKBR3R and BT474R cells Targeting MUC1-C suppresses HER3 phosphorylation (Supplementary Table 1A). For these trastuzumab-resistant cells, HER2 phosphorylates HER3 and disruption of the HER2/HER3 there is no definable IC50 for trastuzumab (Supplementary interaction is associated with HER3 dephosphorylation.5 These Table 1A); accordingly, we used the Bliss independence (BI) findings and the demonstration that targeting MUC1-C method for assessing interactions between two agents when one downregulates HER2 invoked the possibility that MUC1-C of the agents is inactive.37,38 As expected, treatment with inhibition could also affect HER3 activation. Indeed, silencing trastuzumab alone at 40 nM had no effect on SKBR3R cell

Oncogene (2014) 3422 – 3431 & 2014 Macmillan Publishers Limited MUC1-C promotes HER2 activation and trastuzumab resistance D Raina et al 3425

Figure 2. Targeting the MUC1-C CD with GO-203 blocks the interaction with HER2 and suppresses HER2 activation. (a) Schema of the MUC1-C subunit with the 58-aa extracellular domain (ED), the 28-aa transmembrane domain (TM) and the amino-acid sequence of the 72-aa CD. Highlighted is the CQC motif that is necessary and sufficient for dimerization of the MUC1-C subunit. Lysates from SKBR3 (left) and BT474 (right) cells were incubated with the indicated GST or GST-fusion proteins. The adsorbates were immunoblotted (IB) with anti-HER2. Input of the GST proteins was assessed by Coomassie blue staining. (b) D-Amino-acid sequences of the GO-203 and CP-2 peptides. SKBR3 (left) and BT474 (right) cells were treated with 5 mM GO-203 or CP-2 for 48 h. Anti-MUC1-C precipitates were immunoblotted with anti-HER2 and anti- MUC1-C. (c) SKBR3 cells were treated with 5 mM GO-203 or CP-2 for 48 h (left) or with 5 mM GO-203 for the indicated times (right). Lysates were immunoblotted with the indicated antibodies. (d) BT474 cells were treated with 5 mM GO-203 or CP-2 for 48 h. Lysates were immunoblotted with the indicated antibodies. (e and f) SKBR3 (e) and BT474 (f) cells were left untreated (Control; CTL) or treated with 5 mM GO-203 for the indicated times. Cell number (mean±s.e. of three determinations) was determined by Trypan blue staining. IP, immunoprecipitated. growth. However, GO-203 alone was effective in inhibiting growth, combination with trastuzumab in the treatment of trastuzumab- and combining GO-203 with 40 nM trastuzumab was more resistant BT474R cells using the BI (Supplementary Figure S2B and effective than either agent alone (Figure 6d, left and right), Supplementary Table 2) and Chou–Talalay (Supplementary Figure indicating that GO-203 reverses resistance to trastuzumab. An S2C and Supplementary Table 1B) methods. These findings observed BI score of 0.84 as compared with 0.46 for the predicted indicate that GO-203 is synergistic with trastuzumab in the BI supported a synergistic interaction (Supplementary Table 2). treatment of trastuzumab-resistant cells. Based on these findings, we used the Chou–Talalay method to further analyze the interaction between GO-203 and trastuzumab. Under these experimental conditions, we confirmed that the Effects of targeting MUC1-C on lapatinib treatment combination of GO-203 and trastuzumab is synergistic in the of trastuzumab-resistant cells treatment of SKBR3R cells (Figure 6e) with the CIo1 Lapatinib is a small-molecule ATP competitor that inhibits (Supplementary Table 1B). GO-203 was also highly effective in phosphorylation of the HER2 intracellular kinase domain and has

& 2014 Macmillan Publishers Limited Oncogene (2014) 3422 – 3431 MUC1-C promotes HER2 activation and trastuzumab resistance D Raina et al 3426

Figure 3. Interaction between MUC1-C and HER2 is upregulated in trastuzumab-resistant breast cancer cells. (a) SKBR3 (left) and SKBR3R 4 (right) cells (1 Â 10 per well) were left untreated or treated with 80 nM trastuzaumb for 72 h. Cell number (mean±s.e. of three determinations) was determined by Trypan blue staining. (b) Lysates from SKBR3 and SKBR3R cells were immunoblotted (IB) with the indicated antibodies. (c) Anti-MUC1-C precipitates from SKBR3 and SKBR3R cells were immunoblotted with the indicated antibodies. (d) BT474 (left) and BT474R 4 (right) cells (1 Â 10 per well) were left untreated or treated with 80 nM trastuzumab for 72 h. Cell number (mean±s.e. of three determinations) was determined by Trypan blue staining. (e) Lysates from BT474 and BT474R cells were immunoblotted with the indicated antibodies. (f) Anti- MUC1-C precipitates from BT474 and BT474R cells were immunoblotted with the indicated antibodies. IP, immunoprecipitated.

reported activity in trastuzumab-resistant cells.7,39–41 In the present human non-HER2-amplified breast cancer cell lines.24 To define studies with SKBR3R cells, the combination of lapatinib and GO-203 the functional significance of this association, the present studies was more effective in inhibiting growth than lapatinib alone were performed on SKBR3 and BT474 breast cancer cells that (Supplementary Figure S3A, left and right). Synergy between lapatinib overexpress HER2 and are dependent on HER2 for proliferation.3 andGO-203wassupportedbyanobservedBIscoreof0.85as As anticipated, we found that MUC1-C associates with HER2 in compared with 0.58 for the predicted BI (Supplementary Table 3). In these HER2-overexpressing cells. Surprisingly, however, our results addition, lapatinib was synergistically active with GO-203 in inhibiting showed that silencing MUC1-C is associated with the growth of BT474R cells (Supplementary Figure S3B (left and right) and downregulation of HER2 activation and loss of clonogenic Supplementary Table 3). Based on these observations, we assessed survival, supporting the contention that MUC1-C is of the effects of (i) lapatinib alone, (ii) lapatinib þ trastuzumab and importance to HER2 signaling. The MUC1-C subunit includes a (iii) lapatinib þ trastuzumab þ GO-203.UsingSKBR3Rcells,the CD that contains a CQC motif, which is necessary for the formation lapatinib þ trastuzumab combination was not significantly different of MUC1-C homodimers.30 Our results show that the MUC1-C CD is in inhibiting growth as compared with lapatinib alone (Supplementary sufficient to form complexes with HER2 and that this association is Figure S4A, left and right). However, the triple lapatinib þ abrogated by altering the CQC motif to AQA, indicating that trastuzumab þ GO-203 combination was significantly more effective MUC1-C homodimerization is necessary for the MUC1-C/HER2 than lapatinib alone or lapatinib þ trastuzumab (Supplementary interaction. Cell membrane-penetrating peptides, such as GO-203, Figure S4A, left and right) and as evidenced by an observed BI of block homodimerization of endogenous MUC1-C through binding 0.81 compared with a predicted BI of 0.59 (Supplementary Table 4). to the CQC motif.31,32 The present results demonstrating that These findings with the lapatinib þ trastuzumab þ GO-203 combina- treatment of HER2-overexpressing breast cancer cells with GO-203 tion were further supported in studies of BT474R cells (Supplementary blocks the interaction between MUC1-C and HER2 provided Figure S4B, left and right); Supplementary Table 4), indicating that GO- further support for the premise that MUC1-C homodimerization is 203 potentiates the effects of both lapatinib and trastuzumab in necessary for forming complexes with HER2. In addition and as trastuzumab-resistant cells. found with MUC1-C silencing, targeting MUC1-C with GO-203 treatment was associated with marked decreases in HER2 phosphorylation. Overexpression of HER2 in human breast DISCUSSION cancer cells facilitates the formation of HER2/HER3 heterodimers, MUC1-C is a previously unrecognized effector of HER2 activation activation of HER2 and HER2-mediated phosphorylation of HER3.4 Previous work demonstrated that MUC1 associates with HER2 in The importance of HER3 in the HER2/HER3 heterodimer is the mammary glands of an MUC1 transgenic mouse model and in supported by the demonstration that loss of HER3 function

Oncogene (2014) 3422 – 3431 & 2014 Macmillan Publishers Limited MUC1-C promotes HER2 activation and trastuzumab resistance D Raina et al 3427

Figure 4. Downregulation of MUC1-C suppresses HER2 activation in trastuzumab-resistant cells. (a and b) SKBR3R (a) and BT474R (b) cells were infected with lentiviruses expressing a control shRNA (CshRNA) or MUC1shRNA. Lysates were immunoblotted (IB) with the indicated antibodies. (c and d) SKBR3R (c) and BT474R (d) cells were treated with 5 mM GO-203 for the indicated times. Anti-MUC1-C precipitates were immunoblotted with the indicated antibodies. (e and f) SKBR3R (e) and BT474R (f) cells were treated with 5 mM GO-203 for the indicated times. Lysates were immunoblotted with the indicated antibodies. IP, immunoprecipitated. attenuates HER2-mediated transformation.3 In addition to the expression.42 However, the present results show that the downregulation of HER2, we found that targeting MUC1-C is development of trastuzumab resistance over 18 months has associated with suppression of HER3 activation. These findings little if any effect on MUC1-C abundance in SKBR3R and BT474R support a previously unrecognized model in which MUC1-C cells. The basis for this discrepancy in the findings is not clear. homodimerization contributes to HER2 activation and HER3 Nonetheless, the present results demonstrate that trastuzumab phosphorylation. resistance is associated with a substantial (B20-fold) increase in the association of MUC1-C and HER2. Trastuzumab disrupts ligand-independent HER2/HER3 interactions in HER2- Targeting MUC1-C is a potential strategy for circumventing overexpressing cells.5 Our results indicate that targeting MUC1-C trastuzumab resistance attenuates HER2 activation in trastuzumab-resistant cells. These Patients with HER2-overexpressing breast cancers frequently findings raised the possibility that MUC1-C may contribute to display primary unresponsiveness or develop acquired resistance trastuzumab resistance by promoting HER2-mediated signaling. to trastuzumab therapy. Trastuzumab resistance has been Indeed, targeting MUC1-C was associated with downregulation of attributed to the number of mechanisms, including activation of (i) p-HER3 and p-AKT levels, and (ii) phosphorylation of p27, an the phosphatidylinositol-3 kinase and SRC pathways.12,13 Selection AKT substrate. In addition, targeting MUC1-C resulted in decreases for growth of BT474 cells in the presence of trastuzumab for 8 in cyclin E abundance, a finding consistent with the weeks has been associated with the upregulation of MUC1 demonstration that cyclin E levels decrease upon HER2

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Figure 5. Targeting MUC1-C in trastuzumab-resistant cells suppresses HER3 activation and inhibits growth and clonogenic survival. (a) Lysates from the indicated SKBR3R (left) and BT474R (right) cells were immunoblotted (IB) with anti-p-HER3 and anti-HER3. (b) The indicated SKBR3R (left) and BT474R (right) cells were seeded at 4000 and 1000 cells per well, respectively, and incubated for 12 days. The colony number is expressed as the mean±s.e. of three replicates. (c) SKBR3R cells were treated with 5 mM GO-203 for the indicated times. Lysates were immunoblotted with the indicated antibodies. (d) SKBR3R cells were left untreated (Control; CTL) or treated with 5 mM GO-203 for the indicated times. Cell number (mean±s.e. of three determinations) was determined by Trypan blue staining. (e) BT474R cells were treated with 5 mM GO-203 for the indicated times. Lysates were immunoblotted with the indicated antibodies. (f) BT474R cells were left untreated (Control; CTL) or treated with 5 mM GO-203 for the indicated times. Cell number (mean±s.e. of three determinations) was determined by Trypan blue staining. (g) BALB/c nu/nu mice were injected subcutaneously in the flank with 1 Â 107 BT474R cells. The mice were pair-matched when the tumors were 80–100 mm3. Treatment groups consisted of eight mice injected intravenously with phosphate-buffered saline (vehicle control; diamonds) or 7.5 mg/kg GO-203 (squares) each day for 21 days. There was no weight loss in the two groups. The results are expressed as tumor volume (mean±s.e. for the eight mice in each group).

inhibition.43 The downregulation of p27 phosphorylation could Why would MUC1-C contribute to HER2 signaling in breast cancer also be linked to decreases in cyclin E expression as a result of cells? increased localization of p27 to the nucleus and thereby inhibition Epithelia are single cell layers with apical–basal polarity that of CDK2, which in turn results in cyclin E degradation.35,36,44 separate metazoans from the external environment. As such, Further studies will be needed to address the mechanistic basis for robust defense mechanisms emerged during evolution to protect cyclin E downregulation in GO-203-treated trastuzumab-resistant epithelial integrity. The mucins contribute in part to that defense cells. Nonetheless, as both phospho-p27 and cyclin E have been by forming a protective physical barrier.25 The MUC1-N subunit is linked to trastuzumab resistance,20–22 we treated trastuzumab- shed from the epithelial cell surface into this barrier as a first line resistant cells with GO-203 in combination with trastuzumab. The of defense. In turn, the transmembrane MUC1-C subunit functions observation that GO-203 and trastuzumab are highly synergistic in a subsequent line of defense by signaling stress to the interior lends further support to the premise that MUC1-C contributes to of the epithelial cell to promote repair, growth and survival.25 the trastuzumab-resistant phenotype by promoting HER2/HER3- Importantly in this regard, the response of epithelial cells to stress AKT activation. These findings thus indicate that targeting MUC1-C is associated with loss of apical–basal polarity and activation of an can reverse trastuzumab resistance. In addition, our results HER2-mediated proliferation and survival program.45 With loss of indicate that GO-203 can potentiate the effects of lapatinib and polarity, the apical MUC1-C protein is transiently repositioned over the lapatinib þ trastuzumab combination in the SKBR3R and the cell membrane, allowing it to interact with HER2, which is BT474R models. Translation of these findings with regard to the normally sequestered at the basolateral membrane.24,25 The potential effectiveness of combining GO-203 with trastuzumab present results provide evidence that, in associating with HER2, and/or lapatinib in the clinic will therefore require further study. In MUC1-C contributes to HER2 activation and HER3 this respect, a Phase I trial of GO-203 is presently being completed phosphorylation. Thus, a potential advantage of this interaction for patients with refractory solid tumors and, based on the present with MUC1-C could conceivably be prolonged HER2/HER3 results, this agent may be effective in combination with HER2 signaling in the absence of ligand. The epithelial stress response inhibitors in the setting of trastuzumab-resistant breast cancer. is reversible such that the interaction between MUC1-C and HER2

Oncogene (2014) 3422 – 3431 & 2014 Macmillan Publishers Limited MUC1-C promotes HER2 activation and trastuzumab resistance D Raina et al 3429

Figure 6. Targeting MUC1-C reverses trastuzaumab resistance. (a and b) The indicated SKBR3R (a) and BT474R (b) cells (1 Â 104 per well) were left untreated or treated with 80 nM trastuzumab for 72 h. Cell number (mean±s.e. of three determinations) was assessed by Trypan blue staining. (c) SKBR3 cells were treated with GO-203 alone ( þ ), trastuzumab alone (}) and the combination (X) based on the IC50 values listed in Supplementary Table 1A. Growth inhibition data were analyzed by the method of Chou and Talalay using the CalcuSyn program. The dose– effect curves (left) and median–effect plots (right) are shown using mM concentrations for GO-203 and trastuzumab. The CI values are listed in Supplementary Table 1B. (d) SKBR3R cells were treated with GO-203 alone (closed circles) and in combination with 40 nM trastuzumab (closed squares) for 72 h. Growth inhibition plots (left) were generated by Prism GraphPad software and the BI score (Supplementary Table 2) was calculated as described in the Materials and methods. The percentage growth inhibition (mean±s.e. of three determinations) obtained with 5 mM GO-203 was significantly different from that with 5 mM GO-203 þ 40 nM trastuzumab (right). (e) SKBR3R cells were treated with GO-203 alone ( þ ), trastuzumab alone (}) and the combination (X). Growth inhibition data were analyzed by the method of Chou and Talalay using the CalcuSyn program. The dose–effect curves (left) and median–effect plots (right) are shown using mM concentrations for GO-203 and trastuzumab. The CI values are listed in Supplementary Table 1B. NS, nonsignificant. is transient with return of epithelial cell polarity. By contrast, in cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Reactivity was carcinoma cells with sustained activation of the epithelial– detected with horseradish peroxidase-conjugated secondary antibodies mesenchymal transition, MUC1-C is positioned to constitutively and chemiluminescence. interact with the HER2 complex and promote activation of the HER2 pathway. In this way, breast cancer cells would appear to In vitro binding assay have appropriated and subverted a physiologic stress response to Glutathione S-transferase (GST) and GST-MUC1-CD fusion proteins were support their own growth and survival. Importantly, the interac- prepared and incubated with cell lysates as described.27 Adsorbates to tion between MUC1-C and HER2 has been increased further and glutathione-conjugated beads were analyzed by immunoblotting. subverted to circumvent the growth inhibitory effects of trastuzumab treatment, such that targeting MUC1-C reverses trastuzumab resistance. Colony formation assays Cells were seeded onto six-well plates and incubated for 10–14 days. The cells were then washed and stained with 0.5% crystal violet in 25% methanol. Colonies 425 cells were counted in triplicate wells. MATERIALS AND METHODS Cell culture SKBR3 and BT474 breast cancer cells (ATCC, Manassas, VA, USA) were Trastuzumab-resistant BT474R tumor xenograft model maintained in McCoy’s 5a modified medium and Dulbecco’s modified Four- to 6-week-old BALB/c nu/nu female mice were injected subcuta- Eagle’s medium/Ham F12 (1:1) supplemented with 10% heat-inactivated neously with 1 Â 107 BT474R cells in the flank. When tumors were B80– 3 fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM 100 mm , the mice were pair-matched into control and treatment groups L-glutamine. SKBR3R and BT474R cells were selected for resistance to of eight mice each. Phosphate-buffered saline (control vehicle) or GO-203 trastuzumab as described.33 Cells were infected with lentiviral vectors at 7.5 mg/kg body weight was administered daily by intravenous injection expressing a MUC1 short hairpin RNA (shRNA) (Sigma, St Louis, MO, USA) for 21 days. Tumor volumes were calculated as described.27 or a control scrambled shRNA (CshRNA; Sigma). Cells were treated with GO-203 and CP-2 peptides (AnaSpec, San Jose, CA, USA) as described.27 Trastuzumab (Genentech, San Francisco, CA, USA) was dissolved in sterile Determination of IC50 and synergism apyrogen water (stock solution of 21 mg/ml) and stored at 4 1C. Lapatinib Cells were seeded onto 96-well plates in 100 ml of growth medium at a (Selleck Chemicals, Houston, TX, USA) was dissolved in dimethyl sulfoxide. density of 3000 cells per well. After 24 h, the cells were exposed to drugs in Viability was determined by Trypan blue exclusion. treatment medium for an additional 72 h. Cell viability was assessed using the alamar blue viability assay (Invitrogen, Grand Island, NY, USA). Triplicate wells of each treatment were analyzed and each experiment was carried Immunoprecipitation and immunoblot analysis out three times. The IC50 values were determined by nonlinear regression Cell lysates were prepared as described.31 Soluble proteins were of the dose–response data using Prism 5.0 for Mac OSX (GraphPad immunoprecipitated with anti-MUC1-C (Ab5; Neomarkers, Kalamazoo, MI, Software, La Jolla, CA, USA). The presence or absence of synergism USA) or a control immunglobulin G. The precipitates and lysates not between GO-203 and trastuzumab was determined by the method of subjected to immunoprecipitation were immunoblotted with anti-MUC1-C, Chou and Talalay.46,47 Briefly, cells were exposed to 1:1 ratios of the 1 anti-b-actin (Sigma-Aldrich), anti-p-HER2(Tyr1221/1222), anti-HER2, anti-p- respective IC50 values for GO-203 and trastuzumab at (i) 4 Â IC50, (ii) HER3(Tyr1289), anti-HER3, anti-p-AKT(S473), anti-AKT (Cell Signaling ½ Â IC50, (iii) IC50, (iv) 2 Â IC50 and (v) 4 Â IC50. Cell viability was determined Technology, Danvers, MA, USA), anti-p-p27(T198), anti-p27 and anti- after treatment for 72 h. The CI was calculated to determine the presence

& 2014 Macmillan Publishers Limited Oncogene (2014) 3422 – 3431 MUC1-C promotes HER2 activation and trastuzumab resistance D Raina et al 3430 of synergism (CIo1) or antagonism (CI41) using CalcuSyn software contributes to trastuzumab resistance of breast cancer cells. Cancer Res 2005; 65: (Biosoft, Cambridge, UK). 11118–11128. 17 Shattuck DL, Miller JK, Carraway III KL, Sweeney C. Met receptor contributes to Assessment of drug interaction by Bliss independence trastuzumab resistance of Her2-overexpressing breast cancer cells. Cancer Res 2008; 68: 1471–1477. The BI model was also used to assess the interaction between GO-203 and 18 Huang X, Gao L, Wang S, McManaman JL, Thor AD, Yang X et al. Hetero- trastuzumab in studies of trastuzumab-resistant cells. BI was determined 37,38 trimerization of the growth factor receptors erbB2, erbB3, and insulin-like growth using the equation Ei ¼ (EA þ EB) À (EA Â EB), where EA is the fractional factor-1 receptor in breast cancer cells resistant to herceptin. Cancer Res 2010; 70: inhibition for GO-203 alone and EB is that for trastuzumab alone. Using this 1204–1214. equation, if the experimentally measured inhibition is greater than Ei, then 38 19 Mittendorf EA, Wu Y, Scaltriti M, Meric-Bernstam F, Hunt KK, Dawood S et al. Loss the combination is synergistic. Plots were generated by Prism GraphPad of HER2 amplification following trastuzumab-based neoadjuvant systemic ther- software. apy and survival outcomes. Clin Cancer Res 2009; 15: 7381–7388. 20 Scaltriti M, Eichhorn PJ, Cortes J, Prudkin L, Aura C, Jimenez J et al. Cyclin E amplification/overexpression is a mechanism of trastuzumab resistance in CONFLICT OF INTEREST HER2 þ breast cancer patients. Proc Natl Acad Sci USA 2011; 108: 3761–3766. DK holds equity in Genus Oncology and is a consultant to the company. The other 21 Nahta R, Takahashi T, Ueno NT, Hung MC, Esteva FJ. P27(kip1) down-regulation is authors declare no conflict of interest. associated with trastuzumab resistance in breast cancer cells. Cancer Res 2004; 64: 3981–3986. 22 Lee-Hoeflich ST, Pham TQ, Dowbenko D, Munroe X, Lee J, Li L et al. PPM1H is a p27 phosphatase implicated in trastuzumab resistance. Cancer Discov 2011; 1: ACKNOWLEDGEMENTS 326–337. This work was supported by Grants CA97098 and CA166480 awarded by the 23 Li Y, Yu W-H, Ren J, Huang L, Kharbanda S, Loda M et al. Heregulin targets National Cancer Institute, and by the Susan G Komen for the Cure Grant SAC110046 g-catenin to the nucleolus by a mechanism dependent on the DF3/MUC1 protein. (MS and JB). Mol Cancer Res 2003; 1: 765–775. 24 Kufe D. MUC1-C oncoprotein as a target in breast cancer: activation of signaling pathways and therapeutic approaches. Oncogene 2013; 32: 1073–1081. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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